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  • Protocol
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Snapshot photoacoustic topography through an ergodic relay of optical absorption in vivo


Photoacoustic tomography (PAT) has demonstrated versatile biomedical applications, ranging from tracking single cells to monitoring whole-body dynamics of small animals and diagnosing human breast cancer. Currently, PAT has two major implementations: photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM). PACT uses a multi-element ultrasonic array for parallel detection, which is relatively complex and expensive. In contrast, PAM requires point-by-point scanning with a single-element detector, which has a limited imaging throughput. The trade-off between the system cost and throughput demands a new imaging method. To this end, we have developed photoacoustic topography through an ergodic relay (PATER). PATER can capture a wide-field image with only a single-element ultrasonic detector upon a single laser shot. This protocol describes the detailed procedures for PATER system construction, including component selection, equipment setup and system alignment. A step-by-step guide for in vivo imaging of a mouse brain is provided as an example application. Data acquisition, image reconstruction and troubleshooting procedures are also elaborated. It takes ~130 min to carry out this protocol, including ~60 min for both calibration and snapshot wide-field data acquisition using a laser with a 2-kHz pulse repetition rate. PATER offers low-cost snapshot wide-field imaging of fast dynamics, such as visualizing blood pulse wave propagation and tracking melanoma tumor cell circulation in mice in vivo. We envision that PATER will have wide biomedical applications and anticipate that the compact size of the setup will allow it to be further developed as a wearable device to monitor human vital signs.

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Fig. 1: Schematic of the PATER system (drawn not to scale for clarity).
Fig. 2: The ER and the spatial resolution of PATER.
Fig. 3: PATER of the mouse cortical vasculature in vivo.
Fig. 4: Reconstruction artifacts caused by signals from uncalibrated areas.
Fig. 5: Temporal signatures of the PA signals.
Fig. 6: Blood flushing in and out of a tube.
Fig. 7: PATER of biological dynamics in vivo.

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Data availability

All data generated or analyzed within this study are included in the article and ref. 10. The raw data for Figs. 2 and 3 can be downloaded via the following links: Fig. 2,; Fig. 3, All other raw data are available from the corresponding author upon request.

Code availability

The reconstruction algorithm and data processing methods are described in detail in this protocol. The reconstruction algorithm is provided with this protocol as Supplementary Software 1.


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This work was supported in part by National Institutes of Health grants R01 CA186567 (NIH Director’s Transformative Research Award), R01 NS102213, U01 NS099717 (BRAIN Initiative), R35 CA220436 (Outstanding Investigator Award) and R01 EB028277.

Author information

Authors and Affiliations



L.L. and Y.L. developed the imaging system. L.L., Y.L. and Y.Z. designed and performed the experiments. L.V.W. supervised the study. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Lihong V. Wang.

Ethics declarations

Competing interests

L.V.W. has financial interests in Microphotoacoustics, Inc.; CalPACT, LLC; and Union Photoacoustic Technologies, Ltd., which did not support this work.

Additional information

Peer review information Nature Protocols thanks Miya Ishihara, Guenther Paltauf and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Li, Y. et al. Nat. Photonics 14, 164–170 (2020):

Li, Y. et al. J. Biomed. Opt. 25, 070501 (2020):

Li, Y. et al. Light Sci. Appl. 9, 135 (2020):

Supplementary information

Reporting Summary

Supplementary Software 1

Software for PATER reconstruction

Supplementary Data 1

3D model and parts list for the animal holder

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Li, L., Li, Y., Zhang, Y. et al. Snapshot photoacoustic topography through an ergodic relay of optical absorption in vivo. Nat Protoc 16, 2381–2394 (2021).

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